NcGRA2-RT-PCR to Detect Live Versus Dead Parasites in Neospora caninum-Infected Mice

摘要

In the present work, we optimized a recently established NcGRA2-RT-PCR based on RNA to detect live Neosporacaninum parasites in tissue, and compared the results with the conventional inoculation of diagnostic specimen ontocell culture. C57BL/6 mice were experimentally infected with Nc-1 tachyzoites and subsequently euthanized 6 or 12 dayspost infection (dpi). Selected organs were used to search for parasites by (i) PCR using genomic DNA (gDNA), (ii) PCRusing cDNA and (iii) in vitro inoculation of cell culture. At 6 dpi, Neospora-gDNA was detected in 34 out of 36 organs.Viable parasites were detected in 11 (NcGRA2-RT-PCR) and 15 (in vitro cultivation) out of 36 organs. Comparison ofNcGRA2-RT-PCR and in vitro detection gave a fair agreement (kappa 0.29), whereas comparison of PCR using gDNAand RT-PCR or in vitro detection resulted in a slight agreement (kappa 0.05 and 0.08, respectively) only. At 12 dpi, parasitegDNA was found in 10 out of 36 organs. In 7 of these organs viability of parasites was confirmed with NcGRA2-RTPCRand growth of parasites in cell culture. Comparison of NcGRA2-RT-PCR and in vitro detection gave a substantialagreement (kappa 0.8), whereas comparison of PCR using gDNA and RT-PCR or in vitro detection resulted in a moderateagreement (kappa 0.59 and 0.77, respectively). As NcGRA2-RT-PCR is almost as sensitive as in vitro cultivation in detectinglive parasites, it represents a fast, easy and safe method of viable parasite detection, and thus an attractive alternativeto the in vitro cultivation approach.